In recent years, a novel set of small proteins have been discovered in the genome, called micropeptides, microproteins, or miniproteins. Estimates of the number of these proteins in the genome are as high as several thousands, and due to their small size, they were overlooked in the original studies of the human genome and the vast majority remain uncharacterized. With novel techniques based on ribosome profiling, mass spectrometry and RNA sequencing, several of these proteins have been functionally characterized, and shown to perform essential functions in cells and to have crucial roles in human disease. Thus, it is very likely that micropeptides are involved in many biological and pathological contexts. However, the techniques commonly used to characterize micropeptides may remain unfamiliar to many laboratories. In this Collection, we present detailed protocols and demonstrations of key wet-lab and dry-lab techniques for the discovery and validation of novel micropeptides, which can be applied to a diverse range of cell and tissue samples. We hope that this collection can serve as a starting point for researchers interested in searching for unknown micropeptides which play a role in their area of biology or disease focus.